The long-term goal of this research is to explore the molecular mechanisms that bacteria use for cell-cell communication. Here we propose an integrative structural, chemical, and biological study of a recently identified quorum sensing circuit whose functioning depends on the production and detection of a small molecule signal called autoinducer-2 (AI-2). AI-2, unlike other known autoinducers, has the potential to mediate cell-cell communication among different bacterial species. To develop a molecular understanding of how AI-2 is detected, and how sensory information is transduced to control behavior on a community scale, we plan to carry out in-depth studies that combine synthetic organic chemistry, bacterial genetics, biochemistry, and X-ray crystallography. The proposed aims build on previous efforts by the investigators in which the crystal structure of an AI-2 sensor protein, LuxP, was determined in a complex with AI-2, revealing the bound ligand to be a furanosyl borate diester bearing no resemblance to previously characterized autoinducers. Specifically, the first aim is to use this structural information to design, synthesize, and functionally characterize quorum sensing agonists and antagonists. The second aim is to carry out genetic screens to identify dominant missense alleles of LuxP that are incapable of responding to AI-2. Gain-of-function LuxP mutants that signal in the absence of AI-2, or in the presence of AI-2 analogs, will also be identified. In the third specific aim, binding assays capable of measuring the affinity of AI-2 and AI-2 analogs for LuxP will be developed and used to evaluate the binding properties of wild-type and mutant LuxP proteins. In the fourth aim, X-ray structural studies of apo-LuxP, LuxP bound to quorum sensing agonists and antagonists, and LuxP homologs from other bacterial species will be carried out. It is anticipated that the closely coordinated multidisciplinary approach proposed here will lead to substantial new insights into bacterial quorum sensing via AI-2. These studies may also result in the discovery of compounds useful in the development of novel broad-spectrum antibacterial drugs that target quorum sensing.